Plasma activated ringer composition and method for preparing the same

Abstract

The present invention relates to a plasma-activated ringer composition and a method for manufacturing the same. The present invention relates to a method for manufacturing a plasma-activated ringer composition effective for the treatment of lung cancer, comprising the steps of: a) providing a lactate solution; and b) activating the lactate solution with plasma so as to generate reactive oxygen species (ROS) and reactive chlorine species (RCLS) within the lactate solution.

Description

Technology Field

[0001] The present invention relates to a plasma-activated Ringer's composition and a method for manufacturing the same. Specifically, it relates to a plasma-activated Ringer's lactate (PAL) synthesized using a microfluidic plasma platform and a plasma-activated Ringer's composition and a method for manufacturing the same for use in the prevention or treatment of lung cancer. Background Technology

[0002] Plasma, the fourth state of matter, consists of radicals, free electrons, cations, and anions. Plasma is classified as thermal plasma or non-thermal plasma depending on thermodynamic temperature equilibrium.

[0003] Compared to thermal plasma, which requires high pressure and high power, non-thermal plasma (NTP) is advantageous for biological applications because it operates at low pressure and power and does not reach thermodynamic equilibrium.

[0004] Non-thermal plasma is characterized by an electron temperature higher than that of the gas, and it can effectively induce cancer cell death directly or indirectly. Direct treatment involves applying plasma directly to cancer cells, but it has limitations due to the difficulty of treating internal anatomical sites.

[0005] Meanwhile, lung cancer is continuously increasing worldwide due to a lack of early symptoms. According to the International Agency for Research on Cancer, lung cancer had the highest incidence rate in 2022.

[0006] Lung cancer accounted for 12.4% of all cancer cases, and the mortality rate was very high at 31.5%. Common treatment methods for lung cancer include surgery, chemotherapy, and radiation therapy, but each method is accompanied by various serious side effects.

[0007] Conventional chemotherapy is effective in inhibiting cancer growth and spread by targeting rapidly dividing cancer cells, but it has side effects such as inhibition of blood cell production and organ damage. Meanwhile, it has been reported that various chemotherapy regimens induced ferroptosis in lung cancer cells. Prior art literature

[0008] Chinese Patent Publication CN117982653A (Publication Date: May 7, 2024) The problem to be solved

[0009] The invention was proposed to solve the aforementioned problem, and plasma-activated Ringer's lactate (PAL) was synthesized using a microfluidic plasma platform and utilized for lung cancer treatment, and the mechanism of lung cancer treatment by controlled reactive species (RS) of plasma-activated Ringer's lactate (PAL) was elucidated.

[0010] In particular, reactive oxygen species (ROS) were generated through the reaction between plasma and water, and reactive chlorine species (RCL) were generated through the reaction between plasma and chloride ions.

[0011] Accordingly, the problem that the present invention aims to solve is to provide a plasma-activated ringer composition comprising reactive oxygen species and reactive chlorine species to be effective in the prevention or treatment of lung cancer, and a method for manufacturing the same.

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0013] A method for preparing a plasma-activated ringer composition according to one embodiment of the present invention for achieving the above-mentioned problem is a method for preparing a plasma-activated ringer composition effective for the prevention or treatment of lung cancer, and may include a step of providing a lactate solution and b) a step of activating the lactate solution with plasma so that reactive oxygen species (ROS) and reactive chlorine species (RCLS) are generated within the lactate solution.

[0014] In step a) of the method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the lactate solution may include NaCl, CaCl2, and KCl.

[0015] The above lactate may be potassium lactate, magnesium lactate, calcium lactate, sodium lactate, or a combination thereof.

[0016] A method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention may further include the step of mixing the lactate solution with an inert gas before activating the lactate solution with plasma.

[0017] The above inert gas may be one or more selected from the group consisting of nitrogen, helium, and argon.

[0018] In a method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the lactate solution can be activated into plasma by applying voltage to the mixed lactate solution and the inert gas.

[0019] In the method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the voltage is 5.0 kV to 7.0 kV.

[0020] In the method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the ringer composition may be diluted by 20 to 40%.

[0021] According to an exemplary embodiment of the present invention, a lactate solution is supplied at a flow rate of 50 to 150 μL / min and an inert gas is supplied at a flow rate of 1.0 to 10 sccm and mixed, and a voltage of 5.0 kV to 7.0 kV is applied thereto to activate the lactate solution into plasma.

[0022] In a method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the active oxygen species are hydrogen peroxide (H2O2), hydroxyl radicals (OH ㆍ ) or singlet oxygen, 1 It is O2.

[0023] In a method for preparing a plasma-activated ringer composition according to an exemplary embodiment of the present invention, the active chlorine species are HOCl, OCl - or HCl ㆍ am.

[0024] A plasma-activated ringer composition according to another embodiment of the present invention for achieving the above-mentioned problem is effective for the prevention or treatment of lung cancer, wherein the composition is formed by activating a lactate solution with plasma, and the composition may include reactive oxygen species (ROS) and reactive chlorine species (RCIS).

[0025] The lactate solution of the plasma-activated ringer composition according to an exemplary embodiment of the present invention may include NaCl, CaCl2, and KCl.

[0026] The active oxygen species of the plasma-activated ringer composition according to an exemplary embodiment of the present invention are hydrogen peroxide (H2O2) and hydroxyl radicals (OH). ㆍ ) or singlet oxygen, 1 It is O2.

[0027] The reactive oxygen species of the plasma-activated ringer composition according to an exemplary embodiment of the present invention can induce the expression of BAX, PUMA, or NOXA within cells.

[0028] The active chlorine species of the plasma-activated Ringer composition according to an exemplary embodiment of the present invention are HOCl, OCl - or HCl ㆍ am.

[0029] The active chlorine species of the plasma-activated ringer composition according to an exemplary embodiment of the present invention can damage the mitochondrial membrane, electron transport chain (ETC), and DNA within the lung cancer cells.

[0030] The plasma-activated ringer composition according to an exemplary embodiment of the present invention has an apoptosis or ferroptosis effect on the lung cancer cells. Effects of the invention

[0031] According to the present invention, a plasma-activated ringer composition comprising reactive oxygen species and reactive chlorine species effective for killing lung cancer cells and a method for manufacturing the same may be provided. Brief explanation of the drawing

[0032] Figure 1 schematically illustrates a microfluidic-based plasma generator used for the generation of plasma-activated Ringer lactate solution and the synthesis of active species. Figure 2 shows the effect of applied voltage on a plasma-activated Ringer lactate solution. (A) Thermal map of pH and (B) H2O2 concentration. (C) OH-DMPO( ㆍ OH (asterisk) and OCl-DMPO ( ㆍ ESR spectrum of the adduct (indicated by the OCl circle). (D) 1 O2-TEMPO 1 (Indicated by the O2 triangle) ESR spectrum of the adduct; black, blue, and red represent 5.0, 6.0, and 7.0 kV, respectively. (E) NO2- and NO3 - Concentration analysis. (F) H-NMR analysis, black and blue represent untreated and PAL treated at 6.0 kV, respectively. Figure 3 shows A549 lung cancer cells treated with 30% PAL. (A) Analysis of A549 cell viability after PAL treatment generated at various voltages. (B) Viability of various normal cell lines after PAL treatment. (C) Viability of A549 cells after treatment with H2O2 standard solution, PAL, and a solution containing catalase. (D) Live / Dead analysis evaluating the effect of PAL treatment generated at 6.0 kV. The scale bar represents 100 μm. Figure 4 shows the analysis results of active chlorine species. (A) DPD results obtained at various applied voltages. (B) DPD analysis of PAS containing single-component or component-free Ringer's lactate solution. (C) DPD analysis of PAS containing two components of Ringer's lactate solution. (D) DPD analysis of PAS containing three or four components of Ringer's lactate solution. All solutions were treated using a microfluidic plasma system with an applied voltage of 6.0 kV. (E) FT-IR analysis of Ringer's lactate solution exposed to different applied voltages. The black, blue, and red lines represent 5.0, 6.0, and 7.0 kV, respectively. (F) Cell viability analysis of PAS containing various Ringer's lactate solution components. All solutions were diluted to 30%. Figure 5 shows the results of flow cytometry analysis of A549 cells. (A) Quantification of apoptosis via flow cytometry of Annexin V / 7-AAD stained cells. (B) Statistical analysis of apoptosis rates. (C) Quantification of ferroptosis via flow cytometry of BODIPY 581 / 591 C11 stained cells. (D) Statistical analysis of ferroptosis rates. All solutions were diluted to 30%. Figure 6 shows the results of Western blot analysis of A549 cells. (A) Western blot of apoptosis pathway-related proteins (BAX, cytochrome C, caspase 3). (B) Western blot of ferroptosis pathway-related proteins (GPX4 and ALOX12). Figure 7 shows the mechanism of action of plasma-activated Ringer's lactate solution on cancer cells. Specific details for implementing the invention

[0033] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims. Specific details for implementing the present invention will be described in detail below with reference to the attached drawings. Regardless of the drawings, identical reference numerals refer to identical components, and "and / or" includes each of the mentioned items and all combinations of one or more.

[0034] The terms used herein are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the components mentioned.

[0035] Unless otherwise defined, all terms used in this specification (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0036] Hereinafter, a plasma-activated ringer composition according to an embodiment of the present invention and a method for manufacturing the same will be described. These embodiments are solely for the purpose of illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these embodiments.

[0037] Example 1: Preparation of Materials and Experimental Method

[0038] 1) Matter

[0039] Human lung cancer cell line (A549), normal lung epithelial cell line (NHBE), normal breast epithelial cell line (MCF-10A), normal skin keratinocyte cell line (HACAT), normal umbilical cord blood endothelial cell line (HUVEC), and normal colon epithelial cell line (Caco-2) were purchased from ATCC.

[0040] Cells were cultured in Roswell Park Memorial Laboratory (RPMI-1640), Dulbecco Modified Aigle Medium (DMEM), or Dulbecco Modified Aigle Medium F12 (DMEM / F12). All of the above materials were purchased from Invitrogen Gibco (Carlsbad, CA, USA).

[0041] The airway epithelial cell basal medium and bronchial / tracheal epithelial cell growth kit were purchased from ATCC, and the endothelial cell medium and endothelial cell growth supplement were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA).

[0042] Horse serum, human HB-EGF, hydrocortisone, and insulin were purchased from ThermoFisher Scientific (Cleveland, OH, USA), and cholera toxin was purchased from List Biological Labs (Campbell, CA, USA).

[0043] To analyze the active species (RS) generated by PAL, a peroxide analysis kit, DMPO (5,5-dimethyl-1-pyrroline N-oxide), TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl), a nitrate / nitrogen oxide analysis kit, deuteride, and hydrogen peroxide solution were used.

[0044] All products were purchased from ThermoFisher Scientific (Cleveland, OH, USA), and the DPD 1 & 3 test kits for RClS analysis were purchased from Palintest (Gateshead, US).

[0045] Catalase (CAT), sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), sodium lactate (C3H5NaO3), doxorubicin (DOX), elastin, Z-VAD-FMK, deferoxamine (DFO), and ferrosatin-1 (fer-1) were purchased from ThermoFisher Scientific (Cleveland, OH, USA) and used to analyze the active species (RS) generated by PAL.

[0046] Live / dead cell analysis solution was purchased from DyneBio (South Korea), FITC Annexin V Apoptosis Detection Kit containing 7-AAD was purchased from Cytek Biosciences (Fremont, CA, USA), and BODIPY 581 / 591 C11 was purchased from Invitrogen Gibco (Carlsbad, CA, USA).

[0047] For Western blot, RIPA buffer, protease and kinase inhibitors, BCA protein assay kit, anti-BAX antibody, anti-caspase-3 antibody, anti-cytochrome C antibody, anti-β-actin antibody, anti-glutathione peroxidase 4 antibody (GPX4), anti-4 hydroxynonenal antibody (4-HNE), and anti-12 lipoxygenase / ALOX12 antibody were purchased from Abcam (Cambridge, UK).

[0048] 2) Preparation of various cell lines

[0049] A549 cell lines were cultured in RPMI-1640 medium containing 10% FBS and 1% P / S, and NHBE cell lines were cultured in airway epithelial cell basal medium with the addition of a bronchial / tracheal epithelial cell growth kit.

[0050] MCF-10A cell lines were cultured in DMEM / F12 medium containing 5% horse serum, 20 ng / mL human HB-EGF, 0.5 μg / mL hydrocortisone, 10 μg / mL insulin, 100 ng / mL cholera toxin, and 1% P / S. HACAT cell lines were cultured in DMEM medium containing 10% FBS and 1% P / S. HUVEC cell lines were cultured in endothelial cell medium containing endothelial cell growth supplements. Finally, Caco-2 cell lines were cultured in DMEM / F12 medium containing 10% FBS and 1% P / S. All cell lines were maintained at 37°C in a humidity-controlled incubator containing 5% CO2.

[0051] 3) Method for synthesizing plasma-activated Ringer's lactate solution

[0052] A plasma-activated Ringer's lactate (PAL) composition was produced using a microfluidic plasma system (Fig. 1).

[0053] The solution was supplied to the PTFE tube at a constant flow rate (100 μL / min) using a syringe pump. The Ringer's fluid consisted of NaCl (6 g / L), CaCl₂ (0.27 g / L), KCl (0.3 g / L), and CH₃CH(OH)CO₂Na (3.1 g / L).

[0054] The helium gas supply was controlled to 5.0 sccm in the PTFE tube using a mass flow meter and a mass flow regulator.

[0055] The solution and helium combined at the T-junction to form a helium-floating droplet, after which the mixture flowed into the plasma application area. The plasma power supply generated a PAL by applying a voltage between 5.0 kV and 7.0 kV through a copper wire connected to a PTFE tube.

[0056] 4) Characterization of reactive oxygen and nitrogen species

[0057] The pH of PAL was measured five times using a pH meter (Mettler Toledo, Columbus, OH, USA). The H2O2 concentration in the 20-fold diluted PAL samples was measured using a Peroxide Assay Kit (ThermoFisher Scientific, Cleveland, OH, USA) according to the manufacturer's instructions.

[0058] nitrite (NO2 - ) and nitrate (NO3 - The concentration was determined by measuring the absorbance at 540 nm using the Nitrite / Nitrate Assay Kit according to the manufacturer's instructions.

[0059] Hydroxylation (hydroxyl, OH) in PAL ㆍ) and hypochlorite (hypochlorite, OCl ㆍ The presence of radicals was confirmed by electron spin resonance (ESR) spectroscopy using the EMXplus-9.5 / 12 / P / L system (Bruker, Karlsruhe, Germany).

[0060] DMPO was added at a concentration of 20 μg / mL for the detection of DMPO-OH and DMPO-OCl radical adduct complexes.

[0061] singlet oxygen 1 The presence of O₂ was confirmed by ESR spectroscopy by detecting the formation of TEMPO adduct complexes after adding TEMP at a concentration of 2 μg / mL.

[0062] For H-NMR analysis, PAL was rapidly frozen and then freeze-dried for 12 hours (EYELA, Tokyo, Japan). The samples were dissolved in D2O and analyzed by NMR (NMR 400; Agilent 400-MR, Agilent Technologies, Santa Clara, CA, USA).

[0063] 5) Analysis of active chlorine species

[0064] PAL was produced by treating Ringer's lactate solution with plasma at a voltage between 5.0 kV and 7.0 kV.

[0065] The plasma-treated solution was produced by preparing each of the four components of Ringer's lactate solution (NaCl, KCl, CaCl₂, C3H5NaO₃) and exposing them to plasma at 6.0 kV.

[0066] Free chlorine concentration was analyzed using a DPD analysis kit, and absorbance was measured at 535 nm using a UV-Vis-NIR spectrophotometer (Jasco Corporation, Tokyo, Japan). Chlorine concentration was measured using an FT / IR spectrometer (Jasco Corporation, Tokyo, Japan).

[0067] 6) Statistical analysis of cell viability

[0068] The effect of PAL on the survival rate of lung cancer cells and normal cells was measured using WST-8 analysis.

[0069] Cells were cultured in a 96-well plate and then cultured in a CO2 incubator at 37°C for 24 hours.

[0070] The treated solutions included Ringer's fluid, PAL treated at different voltages, PAL treated with catalase, an aqueous solution of the same concentration as the H2O2 generated from PAL, and an H2O2 solution treated with catalase. Each solution was diluted with a medium and treated in 200 μL aliquots for 24 hours.

[0071] After removing the solution, 100 μL of medium containing 10 μL of WST-8 reagent was added, and the cells were cultured in a CO₂ incubator for 1 hour.

[0072] Absorbance was measured at 450 nm using a microplate reader.

[0073] 7) Fluorescence-activated single-cell sorting analysis

[0074] Apoptosis and ferroptosis were evaluated using the FITC Annexin V Apoptosis Detection Kit, 7-AAD, and BODIPY™ 581 / 591 C11, respectively.

[0075] Simply put, A549 cells were seeded into a 6-well plate and cultured in a CO₂ incubator at 37°C for 24 hours.

[0076] Then, cells were treated for 24 hours with 2 mL of PAL, DOX, Elastin, Z-VAD, and PAL generated at 6.0 kV containing DFO at various voltages.

[0077] Finally, the stained cells were counted using an Attune CytPix Acoustic Focusing Cytometer (ThermoFisher Scientific, Cleveland, OH, USA).

[0078] Experimental Example 1: Production of Plasma-Activated Ringer Composition

[0079] The synthesis of plasma-activated Ringer's lactate (PAL) via a non-thermal plasma (NTP) platform focused primarily on the analysis of generated ROS and RNS. The Ringer's composition is based on a lactate solution.

[0080] Since the microfluidic plasma system is a closed system, various parameters such as liquid characteristics (type and flow rate), plasma settings (applied voltage and number of channels), and gas conditions (type and flow rate) can be precisely controlled (Fig. 1).

[0081] Therefore, using this setting enables a detailed analysis of reactive species (RS) within PAL, including variables such as reactive oxygen species (ROS), RNS, and pH.

[0082] Initially, the pH of the PAL generated using various applied voltages decreased dramatically from pH 6.17 to pH 3.50 as the voltage increased (Fig. 2A). This was found to be due to the generation of H2O2, a strong acid. Such low pH disrupts intracellular pH homeostasis and leads to cell damage caused by oxidative stress, which may limit biological applications.

[0083] To overcome this, PAL was diluted to 20 to 40% using a medium, thereby increasing the pH to a range of 6.19 to 7.30 suitable for biomedical applications.

[0084] Hydrogen peroxide (H2O2), hydroxyl radical (OH ㆍ ), singlet oxygen, 1 Various types of ROS, such as O2, were also analyzed in PAL.

[0085] The H2O2 concentration increased with increasing voltage, peaking at 746 μM at a voltage of 7.0 kV (Fig. 2B). Controlling the H2O2 concentration is essential when the goal is to induce cancer cell death.

[0086] For example, cells can maintain homeostasis and regulate responses at low H2O2 concentrations, but at high concentrations, cell damage, senescence, and apoptosis may be induced.

[0087] In addition, an H2O2 concentration of 746 μM killed normal cells and cancer cells. Therefore, PAL was diluted by 20% to 40% to lower the H2O2 concentration to between 83 and 305 μM. That is, it is a level that selectively kills cancer cells.

[0088] Meanwhile, OH ㆍ and 1 The concentration of other ROS, such as O₂, also increased with voltage (Figs. 2C and D).

[0089] Conversely, nitrite (NO2 - ) and nitrate (NO3 - Various types of reactive nitrogen species (RNS), including ) were not detected in PAL (Fig. 2E). This means that PAL does not cause cell death by RNS-mediated apoptosis or necrosis.

[0090] This result shows that factors other than ROS contributed to PAL-induced cell death.

[0091] H-NMR analysis showed that lactic acid was converted to acetic acid in PAL, and no other conversions were observed (Fig. 2F).

[0092] In Ringer's lactate solution, lactic acid is converted by plasma into acetate, glyoxylate, pyruvate, and 2,3-dimethyltartrate, which suggests that 2,3-dimethyltartrate is a major causative agent of cancer cell death.

[0093] However, cancer cell death due to plasma transformation by lactic acid was not expected, as only acetate was detected.

[0094] Experimental Example 2: Selective treatment of lung cancer cells using a plasma-activated Ringer composition (plasma-activated Ringer lactate solution, PAL)

[0095] PAL generated using a microfluidic plasma system effectively induced apoptosis in A549 lung cancer cells without adversely affecting normal cells.

[0096] According to the cell viability results, PAL synthesized at voltages above 5.5 kV induced significant apoptosis under 30% dilution conditions, and at voltages above 6.0 kV, IC 20 A value was observed (Fig. 3A).

[0097] IC when treated with PAL at 20% dilution 20 The values ​​were similar, but were significantly different only at 7.0 kV, and the increase in cell death was not significant.

[0098] In contrast, normal cells (NHBE, MCF-10a, HACAT, HUVEC, Caco-2) consistently maintained a viability of over 90% even after treatment with 20% or 30% diluted PAL produced at 6.0 kV (Fig. 3B). Normal cells had lower oxidation levels than cancer cells and were not negatively affected by PAL under these conditions. Therefore, 30% PAL synthesized at 6.0 kV was selected as the optimal condition for selective cancer cell treatment.

[0099] A cell death assay was performed to evaluate the ability of PAL to kill cells (Fig. 3D). Dead A549 cells (labeled in red) were observed after treatment with 20% or 30% PAL generated at 6.0 kV or higher, but no cell death was observed after treatment with 20% PAL generated at 5.0 kV.

[0100] H2O2-induced cell death, one of the major causes of cancer cell death caused by RS, was analyzed to identify a substance that induces A549 cell death in 30% PAL (Fig. 3C).

[0101] A standard H₂O₂ solution with the same peroxide concentration as 30% PAL generated at 6.0 kV was used as a positive control.

[0102] Catalase was added to the two solutions to compare A549 cell apoptosis. Catalase is H ₂ O ₂ It decomposes into water and oxygen to remove H2O2. No cell death was observed in the catalase-treated solution, which confirms that H2O2 is a major cause of cell death in PAL.

[0103] However, when PAL and H2O2 standard solutions were applied to A549 cells, PAL induced 20% more cell death than the standard solution. Similar results were observed with 20% PAL. These results suggest that other RS ​​contributed to cancer cell death induced by PAL.

[0104] Experimental Example 3: Analysis of lung cancer cell death induced by reactive chlorine species (RCLS)

[0105] HOCl / OCl - , chlorine dioxide (ClO₂), chlorine radical (Cl ㆍ ), and dichlorine radical (Cl2 ㆍ- RClS, such as ), plays an important role in oxidation and disinfection.

[0106] Cl ㆍ is OH ㆍIt reacts more quickly and can be used to effectively target cellular components.

[0107] In addition, HOCl is H2O2 and nitric peroxide (ONOO - It was found to induce a higher level of mitochondrial oxidation reaction than ). Therefore, RClS was analyzed for PAL generated at various voltages using a DPD analysis kit (Fig. 4A).

[0108] A light magenta color was observed at 5.0 kV and 5.5 kV, and a darker magenta color appeared at 6.0 kV or higher, and the UV-Vis-NIR spectrophotometric absorbance measured at 535 nm increased with voltage, indicating that the RClS concentration increased.

[0109] RClS was further analyzed to determine which component among NaCl, KCl, CaCl₂, or C3H5NaO₃ contained in Ringer's lactate affects RClS formation (Figs. 4B-4D).

[0110] A total of 16 solutions containing various combinations of these compounds were prepared, and plasma-activated solutions (PASs) were generated using a microfluidic plasma system at a voltage of 6.0 kV.

[0111] The results showed consistently high concentrations of free chlorine in the solution containing NaCl regardless of the presence of other components (Figs. 4C, 4D), which was explained by the fact that the concentration of NaCl in the Ringer lactate solution is more than 10 times higher than that of KCl or CaCl₂. RClS generated in PAL was confirmed using FT-IR analysis (Fig. 4E).

[0112] 820cm -1 A strong O-Cl absorption peak was detected, indicating that the chlorine in PAL is OCl - Or it was found to exist in the form of HOCl.

[0113] C3H5NaO₃ The PAS without [this] had a lower pH, but all 16 samples showed neutral pH values ​​after being diluted to 30%, and H ₂ O ₂ The concentration was similar.

[0114] The results showed that the A549 cell death rate was significantly higher in PAS containing NaCl than in PAS without NaCl (Fig. 4F), which indicates that NaCl chlorine reacts with plasma to produce OCl - It generates ROS and HOCl, suggesting that this increased A549 cell death. Therefore, it can be said that A549 lung cancer cell death induced by PAL was mainly mediated by ROS and RClS.

[0115] Experimental Example 4: Apoptosis and ferroptosis of lung cancer cells induced by plasma-activated Ringer composition (plasma-activated Ringer lactate solution, PAL)

[0116] PAL induces two major cell death pathways, apoptosis and ferroptosis, in A549 lung cancer cells.

[0117] Lung cancer cells are characterized by inducing iron accumulation by inhibiting iron release or promoting iron absorption.

[0118] Apoptosis is an established cell death pathway mediated by a caspase-dependent mechanism, whereas ferroptosis is a recently identified pathway induced by iron accumulation and lipid peroxidation, leading to cell death through cell membrane damage caused by oxidative stress.

[0119] The pathways of apoptosis and ferroptosis were investigated, respectively, through flow cytometry analysis after Annexin V-FITC / 7-AAD and BODIPY 581 / 591 C11 staining.

[0120] As a result of the apoptosis analysis, the initial apoptosis rate (Annexin V-FITC(+) / 7-AAD(-)) increased to 59% when the voltage was increased to 7.0 kV (Figs. 5A, 5B).

[0121] There was no significant change at 5.5 kV or lower, but at 6.0 kV or higher, it showed a pattern similar to the results observed in the 20μM DOX-treated positive control group.

[0122] Conversely, when A549 cells treated with PAL generated at 6.0 kV were co-treated with the apoptosis inhibitor Z-VAD, the initial apoptosis rate decreased to 12.8%, suggesting that PAL induces apoptosis and promotes cell death in A549 cells.

[0123] As a result of ferroptosis analysis, PAL increased the lipid peroxidation rate to 94.5% due to the emission peak increased by voltage (Fig. 5C, 5D).

[0124] At 6.0 kV or higher, the tendency for cell death induced by PAL was similar to the results observed in 10 m Elastian (positive control for ferroptosis).

[0125] Conversely, when PAL generated at 6.0 kV was co-treated with the ferroptosis inhibitor DFO, the lipid peroxidation rate decreased to 10.1%.

[0126] These results show that PAL induced both apoptosis and ferroptosis in A549 cells.

[0127] The viability of A549 cells was also analyzed after generating PAL at 6.0 kV and co-treating them with the apoptosis inhibitor Z-VAD or the ferroptosis inhibitor Fer-1 and DFO.

[0128] The results showed that cell viability was 15% higher after co-treatment with DFO, indicating that PAL induces ferroptosis more than apoptosis.

[0129] In addition, when comparing Fer-1 and DFO, DFO increased cell viability by 30% and showed a stronger inhibitory effect on ferroptosis induced by PAL than Fer-1, which suggests that targeting iron accumulation is more effective in alleviating ferroptosis in A549 cells solely through the inhibition of lipid peroxidation.

[0130] Since DFO acts as an iron inhibitor and Fer-1 acts as a lipid peroxidation inhibitor, these results indicate that ferroptosis induced by PAL was driven primarily by iron accumulation rather than lipid peroxidation.

[0131] Experimental Example 5: Mechanism of A549 Lung Cancer Cell Death

[0132] Western blot analysis was performed to investigate the mechanism of A549 cell death induced by PAL. Initially, PAL treatment increased intracellular levels of Bax, cytochrome C, and caspase-3, indicating the induction of apoptosis (Fig. 6A).

[0133] In addition, GPX4 levels decreased and ALOX12 marker levels increased, confirming that ferroptosis caused by lipid peroxidation occurred (Fig. 6B).

[0134] These results indicate that PAL induces a combination of apoptosis and ferroptosis in A549 cells (Fig. 7).

[0135] The major substances that generate this death pathway are plasma-induced ROS and RClS, which cause mitochondrial oxidation. ROS activate nuclear p53, inducing the expression of pro-apoptotic factors such as BAX, PUMA, and NOXA.

[0136] BAX moves into mitochondria, damages the outer mitochondrial membrane through oligomerization, and forms holes in the membrane.

[0137] Meanwhile, RClS damages mitochondrial membranes, electron transport chains (ETCs), and DNA, increasing oxidative stress.

[0138] In particular, RClS increases membrane permeability and interferes with ATP synthesis by oxidizing components of electron transport complex I, III, and IV.

[0139] These combined effects lead to the release of cytochrome C from mitochondria, the formation of apoptosomes, and the activation of caspase-9, caspase-7, and caspase-3, which ultimately induces apoptosis.

[0140] In addition, ROS is Fe accumulated in A549 lung cancer cells 2+ It reacts with to induce the Fenton reaction and the Haber-Weiss reaction, which generate OH radicals.

[0141] These radicals oxidize polyunsaturated lipids and lipid peroxides generated by ALOX to form lipid-ROS, causing oxidative damage to cell membranes and inducing ferroptosis.

[0142] This dual mechanism, involving apoptosis and ferroptosis, highlights the therapeutic potential of PAL, particularly in terms of inducing ferroptosis in cancer cells resistant to conventional apoptosis-based therapies.

[0143] In the present invention, plasma-activated Ringer's fluid (PAL) was synthesized using a microfluidic plasma platform, and it was confirmed that apoptosis was induced in A549 cells treated with PAL by H2O2 and HOCl, and that Fenton reaction-based ferroptosis induced by H2O2 occurred.

[0144] Interestingly, PAL induced more cell death than hydrogen peroxide solution, which was found to be due to the presence of active chloroma (RClS). PAL appears to have potential to be developed as a next-generation anticancer treatment capable of killing lung cancer cells without side effects.

[0145] PAL synthesized using a microfluidic-based plasma system can enable the controlled generation of ROS and RClS and suppress RNS formation.

[0146] This control was achieved by using a closed-loop microfluidic plasma system to block atmospheric inflow and generating PAL in a Ringer lactate solution containing oxygen and chlorine-based compounds.

[0147] It was found that PAL generated at 6.0 kV and diluted to 30% effectively induced apoptosis in A549 lung cancer cells without adversely affecting normal lung cells, including normal lung cells and normal lung epithelial cell lines.

[0148] The present invention represents a significant advancement in the utilization of RS in biomedical research. Furthermore, the devised microfluidic plasma system provides quantitative and qualitative control over RS ​​generation, which is generally not achievable with conventional plasma systems.

[0149] The characteristics of this microfluidic plasma system enable the production of customized PALs for selective anticancer treatment.

[0150] PAL exhibited selective cytotoxicity against lung cancer cells, demonstrating its potential as a therapeutic agent particularly effective in targeting cancer cells resistant to apoptosis-based therapies. This is because it can induce apoptosis and ferroptosis.

[0151] Therefore, the present invention provides valuable insights into future cancer treatment research and methods to develop treatments with minimal side effects.

[0152] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, comprising: a) a step of providing a lactate solution; and b) a step of activating the lactate solution with plasma so as to generate reactive oxygen species (ROS) and reactive chlorine species (RCLS) within the lactate solution. Claim 2 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in step a) above, the lactate solution contains NaCl, CaCl2, and KCl. Claim 3 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer according to claim 2, further comprising the step of mixing the lactate solution with an inert gas before activating the lactate solution with plasma. Claim 4 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 3, a voltage is applied to the mixed lactate solution and the inert gas to activate the lactate solution into plasma. Claim 5 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 4, the voltage is 5.0 kV to 7.0 kV. Claim 6 In claim 1, the active oxygen species are hydrogen peroxide (H2O2), hydroxyl radicals (OH ㆍ ) or singlet oxygen, 1 A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized by being O2. Claim 7 In claim 1, the active chlorine species are HOCl, OCl - or HCl ㆍ A method for preparing a plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized by the following. Claim 8 A plasma-activated ringer composition for the prevention or treatment of lung cancer, wherein the composition is formed by activating a lactate solution with plasma, and the composition comprises reactive oxygen species (ROS) and reactive chlorine species (RCLS). Claim 9 A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 8, the lactate solution contains NaCl, CaCl2, and KCl. Claim 10 In claim 8, the active oxygen species are hydrogen peroxide (H2O2), hydroxyl radicals (OH ㆍ ) or singlet oxygen, 1 A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized by being O2. Claim 11 A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 10, the reactive oxygen species induce the expression of BAX, PUMA, or NOXA within the cell. Claim 12 In claim 8, the active chlorine species are HOCl, OCl - or HCl ㆍ A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized by having the following: Claim 13 A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 12, the active chloroma damages the mitochondrial membrane, electron transport chain (ETC), and DNA within the lung cancer cells. Claim 14 A plasma-activated ringer composition for the prevention or treatment of lung cancer, characterized in that, in claim 8, the composition has an apoptosis or ferroptosis effect on the lung cancer cells.

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